The ultimate success of the "Functional Genomics" approach will depend on generating suitable animal models for genetic disorders that manifest clinical phenotypes with obvious metabolic changes and then developing effective therapeutic approaches to reverse these changes. In order to pursue such a course, we chose to work on Fabry disorder because of its unique nature and the challenges it presents in developing much needed therapeutic approaches. Fabry disease is an X-linked inherited disorder of glycolipid metabolism resulting from deficient activity of the lysosomal enzyme, a-galactosidase (AGA). Neutral glycosphingolipids with terminal a-linked galactosyl moieties, globotriaosylceramide (ceramidetrihexoside, CTH: Gala1-4Gal/1-4Glc/1-1Cer) and galabiosylceramide (Gala1-4Gal/1-1Cer), accumulate in the liver, heart, spleen, kidney, vascular endothelial cells and in plasma of the patients with this disorder. Major disease manifestations include paresthesia in the extremities, corneal dystrophy, angiokeratoma, and occlusive vascular disease of the heart, kidney, and brain, leading to premature mortality. Human AGA cDNA and the genomic clone have been isolated, and mapped. Analysis of the AGA gene in Fabry patients revealed heterogeneous molecular lesions such as point mutations and partial gene rearrangements. There is no specific therapy for Fabry disease. Renal transplantation has been performed in Fabry patients with varying outcomes. Enzyme replacement and somatic gene therapy have potential as effective therapies for lysosomal storage diseases. Efficient expression of the human a-Gal A has been reported in NIH-3T3 cells using bicistronic multidrug-resistant gene (MDR) retroviruses. In vitro correction of enzyme deficits has been demonstrated in fibroblasts derived from Fabry patients using recombinant retrovirus. However, a suitable animal model for Fabry disease is required to evaluate the ex vivo and in vivo potential of these therapies. An appropriate model of Fabry disease would be invaluable for the study of the molecular pathophysiology of this human genetic disease as well as for the development of effective therapeutic strategies. In order to develop a mouse model for Fabry disease by disrupting AGA gene by homologous recombination, we isolated and characterized the mouse AGA gene. We found that the mouse and human AGA genes are highly similar in size, gene organization and nucleotide sequence of the coding regions. A targeting vector was constructed using the genomic clones. This vector carries a 1 kb deletion spanning part of exon III and intron III. Three targeted clones were identified as positive for gene disruption and used for generation of AGA null mice. Although the AGA null mice had no clinical phenotype at 10 weeks of age, EM analysis revealed lipid inclusions with electron dense concentric lamellar structures in the lysosomes of renal tubular cells typical of those seen in patients with Fabry disease. Other cellular components appeared morphologically normal. Using fluorescent-labeled Griffonia (Bandeiraea) simplicifolia lectin which selectively binds to a-D-galactosyl residues, we analyzed kidneys of 10 week-old mice and embryonic fibroblasts by confocal microscopy. This analysis revealed intense fluorescence in the kidneys of the mutant mice indicating significant accumulations of compounds containing a-D-galactosyl residues. Cultured fibroblasts from AGA null mouse embryos also displayed significant accumulation of AGA substrates as reflected by the granular staining with intense fluorescence in the cytoplasmic compartment. Analysis of neutral sphingolipids in liver and kidneys of AGA null mice showed a striking accumulation of CTH. In collaboration with Dr. Michel Gottesman, Laboratory of Cell Biology in NCI, we have demonstrated successful use of two bicistronic retroviruses carrying both human AGA cDNA and MDR1 cDNA to correct the enzyme deficits in fibroblasts. These studies were further extended to assess their potential for in vivo gene therapy. In the recent study, we characterized the progression of the disease with aging, and explored the effects of bone marrow transplantation (BMT) on the phenotype. Our findings suggest that BMT may have a potential role in the management of patients with Fabry disease. Additionally, substrate deprivation and gene therapy approaches to reverse the metabolic defects in these mice have proven to be very encouraging. In collaboration with the Dental Clinic, NIDCR, and NINDS we carried out studies to assess oral and craniofacial characteristics in a cohort of patients with Fabry disease in order to facilitate early recognition of this condition and prompt treatment of its manifestations. Data were collected by means of a standardized questionnaire, clinical examination, panoramic and cephalometric radiographs, and magnetic resonance imaging. All dentate subjects without a history of orthodontic treatment (n=7) had malocclusion. Diastemas, particularly among the anterior teeth, were present in 5 of these 7 patients. Of the remaining patients, two had a history of multiple dental extractions resulting in complete edentulism at age 14 and partial edentulism at age 37, and four patients had a history of orthodontic treatment. Anomalies of dental development and eruption were found in some patients. These included a retained mandibular primary incisor with agenesis of the succedaneous permanent tooth, a second molar impaction and a history of delayed eruption. None of the patients had generalized macroscopic enamel defects at the time of our observation. An unusual enamel surface with macroscopic "horizontal waves" of the maxillary anterior teeth and loss of cervical enamel was noticed in one patient. Additionally, one patient had generalized yellow-brown discoloration of his dentition. Histologic evaluation of an extracted molar showed normal dentin tubular number, structure and orientation, normal cementum and pulpal cells (i.e., without cytoplasmic vacuolization;). This case series has reconfirmed oral and craniofacial findings from previous case reports, and most importantly, it has identified additional features of Fabry disease not previously described. Identification of specific disease manifestations will facilitate early diagnosis and intervention of the patients with Fabry disease. These findings will also prove to be valuable in monitoring effectiveness of emerging experimental therapies to treat this painful and fatal disease. One of the clinical symptoms noted in many patients of Fabry disease is reduced saliva resulting in a dry mouth-like condition. In order to identify molecular effects of AGA deficiency on salivary glands, we have analyzed parotid and submaxillary salivary glands of Fabry mice. Parotid and submaxillary salivary glands were analyzed by histopathology and electron microscopy. Gb3 level was determined by thin layer chromatography. AGA activities in parotid and submaxillary salivary glands were significantly lower (~88 and 91%) when compared to age matched one-year-old wild types. Lipid analysis revealed dramatic increases in Gb3 levels in both parotid and submaxillary glands. None or very little Gb-3 accumulation was seen in the salivary glands of wild-type mice. Electron microscopic analysis confirmed the presence of typical lammellar inclusion bodies of Gb3 within the lysosomes of the salivary glands. Large accumulations of Gb-3 in various salivary gland cell types potentially impair salivary gland function in patients with Fabry disease.